Microfluidic cell culture of patient-derived tumor cell spheroids

ABSTRACT

Provided herein are methods for culturing patient-derived tumor cell spheroids in a three-dimensional microfluidic device. The method comprises mincing primary tumor sample in a medium supplemented with serum; treating the minced primary tumor sample with a composition comprising an enzyme; collecting tumor spheroids having a diameter of 10 μm to 500 μm from the enzyme treated sample; suspending the tumor spheroids in biocompatible gel; and culturing the tumor spheroids in a three dimensional microfluidic device. Methods for identifying an agent for treating cancer and microfluidic devices that allow for the simultaneous exposure of the cultured patient-derived primary tumor cell spheroids to a treatment of choice and to control treatment are also provided.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofInternational Application PCT/US2016/012450, filed Jan. 7, 2016, andentitled “MICROFLUIDIC CELL CULTURE OF PATIENT-DERIVED TUMOR CELLSPHEROIDS” which claims priority under 35 U.S.C. § 119(e) to U.S.provisional application number 62/100,607, filed Jan. 7, 2015 and U.S.provisional application number 62/259,928, filed Nov. 25, 2015, thecontents of each of which are incorporated herein by reference in theirentirety.

GOVERNMENT SUPPORT

This invention was made with government support under K08 CA138918-01A1and R01 CA190394-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States. Thecomplexity of most solid tumors limits the understanding of tumorbiology and the development of new and improved treatments. The lack ofrobust in vitro and in vivo models that enable culture of primary humancancers and the reconstruction of the tumor microenvironment hashampered progress in understanding response to targeted therapeutics inreal time. Most current studies rely on cancer cell line culture onplastic in 2-dimensions, or the cost and labor intensive generation ofpatient-derived xenograft (PDX) models in immunocompromised mice. Tumormodels that more closely reflect the conditions in patients arerequired.

SUMMARY OF THE INVENTION

In some aspects, the present disclosure provides a method for culturingpatient-derived tumor cell spheroids in a three-dimensional microfluidicdevice. The method comprises mincing a primary tumor sample in a mediumsupplemented with serum; treating the minced primary tumor sample with acomposition comprising an enzyme; collecting tumor spheroids having adiameter of 10 μm to 500 μm from the enzyme treated sample; suspendingthe tumor spheroids in biocompatible gel; and culturing the tumorspheroids in a three dimensional microfluidic device.

In some embodiments, the primary tumor sample is frozen in a mediumsupplemented with serum and thawed prior to the mincing.

In some embodiments, the collected tumor spheroids are frozen in afreezing medium and then thawed before suspending in the biocompatiblegel. In some embodiments, the minced primary tumor sample comprisestumor pieces in the size of about 1 mm.

In some embodiments, the tumor spheroids having a diameter of 10 μm to500 μm are collected from the enzyme mix treated sample with the use ofa sieve. In some embodiments, the tumor spheroids having a diameter of40 μm to 100 μm are collected from the enzyme mix treated sample withthe use of a sieve. In some embodiments, the tumor spheroids having adiameter of 10 μm to 500 μm are collected by sieving the enzyme mixtreated sample via 500 μm and 10 μm cell strainers to yield tumorspheroids having a diameter of 10 μm to 500 μm.

In some embodiments, the enzyme is collagenase. In some embodiments, thecomposition comprising the enzyme mix comprises a serum-supplementedculture medium, insulin, a corticosteroid, an antibiotic, collagenaseand optionally a growth factor. In some embodiments, the corticosteroidis hydrocortisone.

In some embodiments, the minced primary tumor sample is treated with thecomposition comprising the enzyme in an amount or for a time sufficientyield a partial digestion of the minced primary tumor sample.

In some embodiments, the minced primary tumor sample is treated with theenzyme mix for 30 minutes to 15 hours at a temperature of 25° C. to 39°C. In some embodiments, the minced primary tumor sample is treated withthe composition comprising the enzyme for 30 minutes to 60 minutes at atemperature of 37° C.

In some embodiments, the biocompatible gel is collagen or BD Matrigel™Matrix Basement Membrane. In some embodiments, the biocompatible gel isa fibrin hydrogel. In some embodiments, the fibrin hydrogel is generatedfrom thrombin treatment of fibrinogen.

In some embodiments, the primary tumor sample is obtained from asubject. In some embodiments, the primary tumor sample is a patientderived xenograft (PDX).

In some embodiments, the three dimensional microfluidic device comprisesone or more fluid channels flanked by one or more gel cage regions,wherein the one or more gel cage regions comprises the biocompatible gelin which the tumor spheroids are embedded, and wherein the devicerecapitulates in vivo tumor microenvironment.

In some embodiments, the three dimensional microfluidic device comprisesa substrate comprised of an optically transparent material and furthercomprising i) one or more fluid channels; ii) one or more fluid channelinlets; iii) one or more fluid channel outlets; iv) one or more gel cageregions; and v) a plurality of posts; wherein all or a portion of eachgel cage region is flanked by all or a portion of one or more fluidchannels, thereby creating one or more gel cage region-fluid channelinterface regions; each gel cage region comprises at least one row ofposts which forms the gel cage region; and the one or more gel cageregion has a height of at least 500 μm. In some embodiments, the one ormore gel cage region has a height of 600 μm, 700 μm, 800 μm, 900 μm, or1000 μm. In some embodiments, the one or more gel cage region has aheight sufficient for at least 200-1000 μm above the tumor cellspheroids.

In some embodiments, the gel cage region has a cuboidal shape. In someembodiments, the device comprises 2 gel cage regions. In someembodiments, a portion of a first gel cage region is flanked by aportion of a second gel cage region, thereby creating a gel cageregion-gel cage region interface region. In some embodiments, the firstand second gel cage regions are separated by a barrier which does notallow intermixing between components present in the two gel cageregions.

In some aspects, the present disclosure provides a method foridentifying an agent for treating cancer. The method comprises culturingpatient-derived tumor cell spheroids in a three-dimensional microfluidicdevice as described herein in the presence and absence of a first testagent and detecting a change in the tumor cell spheroid cultureindicative of a response likely to result in reduction in proliferationand/or dispersion of the tumor cell spheroids in the presence of thefirst test agent as compared to the absence of the first test agent;wherein if the change in the tumor cell spheroid culture is indicativeof a response likely to result in a reduction in the proliferationand/or dispersion of the tumor cell spheroids in the presence of thefirst test agent as compared to the absence of the first test agent,then the first test agent can be used to treat cancer.

In some embodiments the method comprises, culturing patient-derivedtumor cell spheroids in the presence of the test agent comprisesintroducing the test agent into the one or more fluid channels of adevice described herein, wherein the one or more gel cage regions of thedevice comprises a gel in which the tumor spheroids are embedded; andculturing the tumor spheroids under suitable culture conditions.

In some embodiments, the change in the tumor cell spheroid culture isdetected chemically, physically, or a combination thereof. In someembodiments, the change in the tumor cell spheroid culture is detectedvisually. In some embodiments, the proliferation and/or dispersion ofthe tumor cell spheroids is determined using by confocal imaging.

In some embodiments, the change in the tumor cell spheroid culture is adecrease in size and/or number of cells of one or more tumor cellspheroids in the culture.

In some embodiments, the change in the tumor cell culture is detectedchemically. In some embodiments, the change in the tumor cell spheroidculture is determined by detection of the presence of a biologicalmolecule secreted into the culture supernatant. In some embodiments, thebiological molecule is a protein, carbohydrate, lipid, nucleic acid,metabolite, or a combination thereof. In some embodiments, thebiological molecule is a cytokine or a chemokine.

In some embodiments, the method includes obtaining a sample of tumorcell spheroid culture supernatant.

In some embodiments, the method includes detecting a cytokine profile orchemokine profile in the tumor cell spheroid culture supernatant.

In some embodiments, the test agent inhibits epithelial-mesenchymaltransition (EMT).

In some embodiments, the first test agent is a small molecule, a nucleicacid molecule, an RNAi agent, an aptamer, a protein or a peptide, anantibody or antigen-binding antibody fragment, a ligand orreceptor-binding protein, a gene therapy vector, or a combinationthereof.

In some embodiments, the first test agent is a chemotherapeutic agent,an immunomodulatory agent, or radiation. In some embodiments, the firstagent is a chemotherapeutic agent selected from the group consisting ofan alkylating agent, an antimetabolite, an anthracycline, a proteasomeinhibitor, and an mTOR inhibitor.

In some embodiments, the first test agent is an immune modulator. Insome embodiments, the first test agent is an immune checkpointinhibitor.

In some embodiments, the patient-derived tumor cell spheroids arecultured in the presence of a second test agent. In some embodiments,the second test agent is an anti-cancer agent.

In some embodiments, the anti-cancer agent is a chemotherapeutic agent,an immunomodulatory agent, or radiation. In some embodiments, the secondtest agent is an immune modulator. In some embodiments, the second testagent is an immune checkpoint inhibitor.

In embodiments, any one of the first test agents may be combined withany one of the second test agents.

In some aspects, the present disclosure provides a microfluidic device.The device comprises a substrate comprised of an optically transparentmaterial and further comprising i) a first gel cage region and a secondgel cage region; ii) a first fluid channel and a second fluid channel;iii) one or more fluid channel inlets; iv) one or more fluid channeloutlets; and v) a plurality of posts; wherein a portion of the first gelcage region is flanked by a portion of the second gel cage region,thereby creating a gel cage region-gel cage region interface region;wherein the first and second gel cage regions are separated by a barrierwhich does not allow intermixing between components present in the twogel cage regions; wherein a portion of the first gel cage region isflanked by all or a portion of the first fluid channel, thereby creatinga first gel cage region-fluid channel interface region; wherein aportion of the second gel cage region is flanked by all or a portion ofthe second fluid channel, thereby creating a second gel cageregion-fluid channel interface region; and, wherein each gel cage regioncomprises one row of posts along the length of the gel cage region atthe first and second gel cage region-fluid channel interfaces.

In some embodiments, each gel cage region has a height of at least 500μm. In some embodiments, each gel cage region has a height of 600 μm,700 μm, 800 μm, 900 μm, or 1000 μm. In some embodiments, each gel cageregion has a cuboidal shape.

In some aspects, the present disclosure provides a method foridentifying an agent for treating cancer, the method comprising: a)introducing a test agent into the first fluid channel of a device asdescribed herein, wherein each gel cage region of the device comprises agel in which the tumor spheroids are embedded; and b) detecting a signalin the first cage region indicative of a response likely to result in areduction in proliferation and/or dispersion of the tumor cell spheroidsin the first gel cage region as compared that in the second gel cageregion; wherein if the signal in the first cage region is indicative ofa response likely to result in a reduction in the proliferation and/ordispersion of the tumor cell spheroids in the first gel cage region ascompared that in the second gel cage region, then the agent can be usedto treat cancer.

In some aspects, the present disclosure provides a method for treatingcancer in a subject, the method comprising: a) obtaining a tumor samplefrom the subject; b) identifying an agent that can be used to treatcancer in the subject according to a method described herein; and c)administering the agent to the subject.

Each of the embodiments and aspects of the invention can be practicedindependently or combined. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including”, “comprising”, or “having”,“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

These and other aspects of the inventions, as well as various advantagesand utilities will be apparent with reference to the DetailedDescription. Each aspect of the invention can encompass variousembodiments as will be understood.

All documents identified in this application are incorporated in theirentirety herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 shows mesothelioma cells are induced to disperse by co-culturewith human umbilical vein endothelial cells (HUVECs).

FIG. 2 shows co-culture of mesothelioma spheroids with HUVECs.

FIG. 3 shows mesothelioma cell dissemination in 3-D culture over aperiod of 48 hours.

FIG. 4 depicts an exemplary immune response in melanoma spheroidsexposed to anti-PD1 in the absence and presence of anti-CD28co-stimulation.

FIG. 5 depicts an exemplary cytokine profile for aPD1 exposure and αPD1in combination with anti-CD28.

FIG. 6 depicts an exemplary immune profiling of treated samples.

DETAILED DESCRIPTION OF THE INVENTION

One major factor in the lack of success in improving patient prognosisand survival after cancer diagnosis is the limitation of current invitro and in vivo cancer models. The data obtained from studies usingthe current models translates poorly into human clinical practicebecause of their lack of concordance with the situation present in thehuman body (Staton C A, Stribbling S M, Tazzyman S, Hughes R, Brown N J,Lewis C E. Current methods for assaying angiogenesis in vitro and invivo. Int J Exp Pathol 2004; 85:233-48). For example, a Boyden chambertest, commonly used to study the invasive properties of a neoplasticcell population of interest, measures the ability of cells to migrateacross an artificial barrier. However, a neoplastic cell will neverencounter such an artificial barrier in a native environment. Humantumor cells in vivo typically form three-dimensional structures, withdrug, metabolite, and cell-cell interaction kinetics much different fromthose in two-dimensional culture.

While patient-derived xenografts (PDX) represent a significant advanceover traditional cancer cell line-based studies, this model system toohas a number of important limitations. These include the need forexpensive cohorts of immunocompromised mice, a long period time requiredto establish sufficient numbers of tumors, and, because of this problemof scale, a limited ability to test multiple drug concentrations and/orcombinations. These challenges pose a major hurdle for using this systemto match appropriate therapies to individual patients, the overall goalof personalized cancer medicine.

Some aspects of this disclosure address and overcome at least some ofthe shortcomings of the current in vitro and in vivo models of cancerdescribed above. Some aspects of the present disclosure are based on thesurprising discovery that primary tumor specimens can be isolated andgrown in a three-dimensional (3D) microfluidic culture device. Thistechnology enables the culture of individual patient tumors and realtime evaluation of novel therapeutics in an unprecedented fashion. Priorto the instant disclosure, it was not known whether tumor spheroidsobtained from primary tumor would grow in vitro in a 3D microfluidicdevice. In fact, previous attempts to grow micro-dissected tumor samplesin a 3D microfluidic device were unsuccessful. The present disclosureprovides, in some aspects, methods to isolate and culture tumorspheroids from primary human tumors. In some aspects, the technologyrecapitulates the tumor microenvironment, enabling cell-cellinteractions that reflect the endothelial -cancer cell interface, andallowing controlled analysis of growth factor and cytokine mediatedeffects.

In some aspects, the methods described herein enable the analysis ofconditioned media during co-culture of tumor spheroids with endothelialcells, providing a unique opportunity to measure primary tumor cytokineproduction. For example, media exchange every 2 days during spheroidculture provides the opportunity to store conditioned media for analysisof individual cytokines, such as IL-6 by enzyme-linked immunosorbentassay (Zhu et al., Cancer Discov 2014 Apr.; 4 (4):452-65; incorporatedherein by reference in its entirety) or broad cytokine profiling usingluminex multiplex technology, for example (Lash et al., J ImmunolMethods 2006 Feb. 20;309(1-2):205-8; incorporated herein by reference inits entirety). Accordingly, aspects of the present disclosure relate tomethods for culturing patient-derived tumor cell spheroids in athree-dimensional microfluidic device. The method comprises mincing aprimary tumor sample in a medium supplemented with serum; treating theminced primary tumor sample with a composition comprising an enzyme;collecting tumor spheroids having a diameter of 10 μm to 500 μm from theenzyme treated sample; suspending the tumor spheroids in biocompatiblegel; and culturing the tumor spheroids in a three dimensionalmicrofluidic device.

As used herein, the term “tumor” refers to a neoplasm, i.e., an abnormalgrowth of cells or tissue and is understood to include benign, i.e.,non-cancerous growths, and malignant; i.e., cancerous growths includingprimary or metastatic cancerous growths.

Examples of neoplasms include, but are not limited to, mesothelioma,lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer),skin cancer (e.g., melanoma), stomach cancer, liver cancer, colorectalcancer, breast cancer, pancreatic cancer, prostate cancer, blood cancer,bone cancer, bone marrow cancer, and other cancers.

The term “tumor spheroid,” or “tumor cell spheroid” as used herein,refers to an aggregation of tumor cells constituting a small mass, orlump of tumor cells. In some embodiments, tumor spheroids are less thanabout 3 cm, less than about 2 cm, less than about 1 cm, less than about5 mm, less than about 2.5 mm, less than about 1 mm, less than about 100μm, less than about 50 μm, less than about 25 μm, less than about 10 μm,or less than about 5 μm in diameter. In some embodiments, the tumorspheroids have a diameter of 10 μm to 500 μm. In some embodiments, thetumor spheroids have a diameter of 40 μm to 100 μm. In some embodiments,the tumor spheroids have a diameter of 40 μm to 70 μm.

The term “primary tumor sample” as used herein refers to a samplecomprising tumor material obtained from a subject having cancer. Theterm encompasses tumor tissue samples, for example, tissue obtained bysurgical resection and tissue obtained by biopsy, such as for example, acore biopsy or a fine needle biopsy. The term also encompasses patientderived xenograft (PDX). Patient derived xenografts are created whencancerous tissue from a patient's primary tumor is implanted directlyinto an immunodeficient mouse (see, for example, Morton C L, Houghton PJ (2007). “Establishment of human tumor xenografts in immunodeficientmice”. Nature Protocols 2 (2): 247-50; Siolas D, Hannon G J (September2013). “Patient-derived tumor xenografts: transforming clinical samplesinto mouse models”. Cancer Research 73 (17): 5315-9). PDX mirrorspatients′ histopathological and genetic profiles. It has improvedpredictive power as preclinical cancer models, and enables the trueindividualized therapy and discovery of predictive biomarkers.

In some embodiments, the subject is a human. In some embodiments, thesubject is a non-human mammal or a non-human vertebrate. In someembodiments, the subject is laboratory animal, a mouse, a rat, a rodent,a farm animal, a pig, a cattle, a horse, a goat, a sheep, a companionanimal, a dog a cat, or a guinea pig.

In some embodiments, the primary tumor sample is collected in aserum-supplemented medium, for example but not limited to, RPMI mediumsupplemented with 10% fetal bovine serum. The sample is then minced,i.e, cut or chopped into tiny pieces. In some embodiments, the sample isminced on ice. In some embodiments, the minced primary tumor samplecomprises tumor pieces in the size of about 3 mm, 2.5 mm, 2.0 mm, 1.5mm, 1.0 mm, 0.5, or 0.25 mm.

In some embodiments, the primary tumor sample is not frozen and thawed.

In some embodiments, minced primary tumor sample is frozen in a mediumsupplemented with serum and thawed prior to treating with thecomposition comprising the enzyme. In some embodiments, the mincedprimary tumor sample is frozen for at least 6 hours 12 hours, 24 hours,2 days, 1 week or one month. In some embodiments, the minced primarytumor sample is frozen at −80° C. In some embodiments, the mincedprimary tumor sample is frozen in liquid nitrogen. In some embodiments,the minced primary tumor sample is frozen in a medium supplemented withserum. In some embodiments, the minced primary tumor sample is frozen ina mixture containing serum and solvent such as Dimethyl sulfoxide(DMSO). In some embodiments, the minced primary tumor sample is frozenin a mixture containing fetal bovine serum and Dimethyl sulfoxide(DMSO).

In some embodiments, the frozen minced primary tumor sample is thawed,i.e., defrosted, before treating the sample with a compositioncomprising an enzyme. In some embodiments, the minced primary tumorsample is thawed in a water bath kept at about 37° C. (e.g., 35° C., 36°C., 37° C., 38° C., or 39° C.). In some embodiments, the minced primarytumor sample is thawed at room temperature.

The minced primary tumor sample is treated with an enzyme mix to digestthe tumor samples. In some embodiments, the composition comprising anenzyme includes collagenase. In some embodiments, the compositioncomprising an enzyme includes a serum-supplemented culture medium,insulin, one or more corticosteroids, one or more antibiotics,collagenase and optionally one or more growth factors.Serum-supplemented culture media, corticosteroids, antibiotics, andgrowth factors are well-known in the art. In some embodiments, thecomposition comprising an enzyme comprises DMEM or RPMI, fetal bovineserum, insulin, epidermal growth factor, hydrocortisone, Penicillinand/or Streptomycin, and collagenase. In some embodiments, thecomposition comprising an enzyme comprises further comprises a bufferingagent such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES).

“Treating the minced primary tumor sample with a composition comprisingan enzyme” comprises incubating the minced tumor samples with the enzymecomposition for at least 1 hour. In some embodiments, the minced tumorsamples are incubated with the enzyme mix for at least 2 hours, at least4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least12 hours, at least 15 hours or at least 24 hours. In some embodiments,the minced primary tumor sample is incubated with the enzyme mix at 25°C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34°C., 35° C., 36° C., 37° C., 38° C., or 39° C. In some embodiments, theminced primary tumor sample is incubated with the enzyme mix at 37° C.

In some embodiments, the minced primary tumor sample is treated with thecomposition comprising the enzyme in an amount or for a time sufficientyield a partial digestion of the minced primary tumor sample. In someembodiments, the minced primary tumor sample is treated with thecomposition comprising the enzyme for 30 minutes to 15 hours at atemperature of 25° C. to 39° C.

Collecting tumor spheroids from the enzyme mix treated sample comprisescentrifuging and washing the sample at least twice followed by isolatingthe digested tumor spheroids of the desired size. In some embodiments,the enzyme mix treated sample is centrifuged and washed using phosphatebuffered saline (PBS) at least twice. Tumor spheroids of the desiredsize are collected using sieves. In some embodiments, the tumorspheroids having a diameter of 10 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400μm, 450 μm, and 500 μm are collected from the enzyme mix treated samplewith the use of a sieve. In some embodiments, the tumor spheroids havinga diameter of 40 μm to 100 μm are collected from the enzyme mix treatedsample with the use of a sieve. In some embodiments, the tumor spheroidshaving a diameter of 40 μm, 50 μm, 60 μm and 70 μm are collected fromthe enzyme mix treated sample with the use of a sieve.

The tumor spheroids having a desired diameter are collected by sievingthe enzyme mix treated sample through cell strainers. In someembodiments, the tumor spheroids having a diameter of 10 μm to 500 μmare collected by sieving the enzyme mix treated sample via 500 μm and 10μm cell strainers to yield tumor spheroids having a diameter of 10 μm to500 μm. In some embodiments, the tumor spheroids having a diameter of 40μm to 100 μm are collected by sieving the enzyme mix treated sample via100 μm and 40 μm cell strainers to yield tumor spheroids having adiameter of 10 μm to 500 μm. The tumor spheroids of the desired diameterare collected and suspended in a biocompatible gel. Examples ofbiocompatible gel include collagen, BD Matrigel™ Matrix BasementMembrane, or fibrin hydrogel (e.g., fibrin hydrogel generated fromthrombin treatment of fibrinogen).

In some embodiments, the collected tumor spheroids are not frozen andthen thawed before suspending in the biocompatible gel.

In some embodiments, the collected tumor spheroids are frozen in afreezing medium and then thawed before suspending in the biocompatiblegel. In some embodiments, the collected tumor spheroids are frozen forat least 6 hours 12 hours, 24 hours, 2 days, 1 week or one month. Insome embodiments, the collected tumor spheroids are frozen at −80° C. Insome embodiments, the collected tumor spheroids are frozen in liquidnitrogen. In some embodiments, the collected tumor spheroids are frozenat −80° C. overnight, and then transferred to liquid nitrogen forstorage. In some embodiments, the collected tumor spheroids are frozenin a medium supplemented with serum. In some embodiments, the collectedtumor spheroids are frozen in a mixture containing culture medium suchas DMEM or RPMI, fetal bovine serum and solvent such as Dimethylsulfoxide (DMSO). The frozen spheroids are thawed, for example overnightat 4° C., and then suspended in the biocompatible gel.

The tumor spheroids are cultured, i.e., grown, in a three dimensional(3D) microfluidic device. In some embodiments, the tumor spheroids arecultured with endothelial cells, such as human umbilical veinendothelial cells (HUVECs). In some embodiments, the tumor spheroids arecultured with or without endothelial cells for at least 1 day, at least2 days, at least 4 days, at least 6 days, at least 1 week, or at least 2weeks.

3D microfluidic devices are known in the art and include, for example,but not limited to, the devices described in US 2013/0143230, EP2741083,US 2014/0057311, and U.S. Pat. No. 8,748,180, the disclosures of whichare incorporated by reference herein.

In some embodiments, a 3D microfluidic device refers to a device thatcomprises one or more fluid channels flanked by one or more gel cageregions, wherein the one or more gel cage regions comprises thebiocompatible gel in which the tumor spheroids are embedded, and whereinthe device recapitulates, i.e., mimics, the in vivo tumormicroenvironment. In order to facilitate visualization, the microfluidicdevice is typically comprised of a substrate that is transparent tolight, referred to herein as “an optically transparent material”. Aswill be appreciated by those of skill in the art, suitable opticallytransparent materials include polymers, plastic, and glass. Examples ofsuitable polymers are polydimethylsiloxane (PDMS), poly(methylmethacrylate) (PMMA), polystyrene (PS), SU-8, and cyclic olefincopolymer (COC). In some embodiments, all or a portion of the device ismade of a biocompatible material, e.g., the material is compatible with,or not toxic or injurious to, living material (e.g., cells, tissue).

The fluid channel can be used to contain a (one or more) fluid (e.g.,cell culture media), cells such as endothelial cells, cellular material,tissue and/or compounds (e.g., drugs) to be assessed, while the gel cageregions may be used to contain a gel (e.g., biologically relevant gel,such as collagen, Matrigel™, or fibrin hydrogel (e.g., fibrin hydrogelgenerated from thrombin treatment of fibrinogen)). In some embodiments,the 3D microfluidic device comprises the device described in US2014/0057311, the disclosure of which is incorporated by referenceherein. In particular, paragraphs [0056] to [0107] which describe theregions, channels, chambers, posts, and arrangement of posts, andparagraphs [0127] to [0130] which describe the methods of making thedevice are incorporated by reference herein.

The original gel region described in US 2014/0057311, and incorporatedby reference herein, was designed to study biology of individual celllines, which requires a relatively small volume. This small volume isinadequate to capture whole tissue sections (even when micro-dissected).Accordingly, in some embodiments, the gel cage region has a height of500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm. This device canaccommodate>10, >20, >30, >40 and>50 spheroids, which is necessary tocapture the full heterogeneity of the primary tumor. The method of claim18, wherein the one or more gel cage region has a height sufficient forat least 200-1000 μm above the tumor cell spheroids.

In some embodiments, the gel cage region has a cuboidal shape. Thecuboidal shape is better suited to accommodate the shape of primarytissue sections, and concentrates spheroids, which facilitatesextraction after endothelial co-culture, and implantation intoimmune-compromised mice for tumor tissue expansion.

Aspects of the disclosure also relate to a microfluidic device thatallows for the simultaneous exposure of the cultured patient-derivedprimary tumor cell spheroids to a treatment of choice and to controltreatment. Thus, the device provides a built-in internal control. Thedevice comprises a substrate comprised of an optically transparentmaterial and further comprising

i) a first gel cage region and a second gel cage region

ii) a first fluid channel and a second fluid channel;

iii) one or more fluid channel inlets;

iv) one or more fluid channel outlets; and

v) a plurality of posts;

wherein

a portion of the first gel cage region is flanked by a portion of thesecond gel cage region, thereby creating a gel cage region-gel cageregion interface region;

the first and second gel cage regions are separated by a barrier whichdoes not allow intermixing between components present in the two gelcage regions;

a portion of the first gel cage region is flanked by all or a portion ofthe first fluid channel, thereby creating a first gel cage region-fluidchannel interface region;

a portion of the second gel cage region is flanked by all or a portionof the second fluid channel, thereby creating a second gel cageregion-fluid channel interface region; and

each gel cage region comprises one row of posts along the length of thegel cage region at the first and second gel cage region-fluid channelinterfaces.

Because the first and second gel cage regions are separated by abarrier, no intermixing between components present in the two gel cageregions takes place, i.e., the barrier is impermeable to cells, cellularmaterial, molecules secreted by the cells, tissue and/or compounds(e.g., drugs) . In some embodiments, the barrier is made of suitableimpermeable material, such as but not limited to, polydimethylsiloxane(PDMS), poly(methyl methacrylate) (PMMA), polystyrene (PS), SU-8, andcyclic olefin copolymer (COC). The gel-cage-fluid channel interfaces, onthe other hand, are lined by a row of posts and when a gel is present inthe gel cage regions, the gel can be contacted with any fluid present inthe fluid channels.

In some embodiments, the gel cage regions of the device have a height of500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm. In some embodiments,the gel cage regions of the device have a cuboidal shape. In someembodiments, the device is used to culture tumor spheroids using themethods described herein.

It was not known whether tumor spheroids obtained from primary tumorwould grow in vitro in a 3D microfluidic device in the presence of atest agent, e.g., enabling the identification of an agent as ananti-cancer agent. This was particularly the case in connection withsuccessfully culturing tumor spheroids in the presence of immune checkpoint inhibitors. Aspects of the disclosure also include methods for ofidentifying an agent for treating cancer. In some embodiments, themethod for identifying an agent for treating cancer, comprises culturingpatient-derived tumor cell spheroids in a three-dimensional microfluidicdevice as described herein in the presence and absence of a test agent,detecting a change in the tumor cell spheroid culture indicative of aresult likely to yield a reduction in proliferation and/or dispersion ofthe tumor cell spheroids in the presence of the first test agent ascompared to the absence of the first test agent; wherein if the changein the tumor cell spheroid culture is indicative of a result likely toyield reduction in the proliferation and/or dispersion of the tumor cellspheroids in the presence of the first test agent as compared to theabsence of the first test agent, then the first test agent can be usedto treat cancer.

In some embodiments, the method for identifying an agent for treatingcancer , comprises a) introducing a test agent into the first fluidchannel of the device described herein, wherein each gel cage region ofthe device comprises a gel in which the tumor spheroids are embedded;and b) detecting a change in the tumor cell spheroid culture indicativeof a reduction in proliferation and/or dispersion of the tumor cellspheroids in the first gel cage region as compared that in the secondgel cage region; wherein if the change in the tumor cell spheroidculture is indicative of a reduction in the proliferation and/ordispersion of the tumor cell spheroids in the first gel cage region ascompared that in the second gel cage region, then the agent can be usedto treat cancer.

“Culturing patient-derived tumor cell spheroids in the presence of thetest agent” comprises introducing the test agent into the one or morefluid channels of the device described herein, wherein the one or moregel cage regions of the device comprises a gel in which the tumorspheroids are embedded; and culturing the tumor spheroids under suitableculture conditions. Suitable conditions include growing the tumor cellspheroids under standard cell culture conditions (e.g. at 37° C. in ahumidified atmosphere of >80% relative humidity air and 5 to 10% CO₂).

In some embodiments, the tumor spheroids are cultured in the presence ofendothelial cells. In some embodiments, the tumor spheroids are culturedin the presence of endothelial cells for at least 1 day, at least 2days, at least 4 days, at least 6 days, at least 1 week, or at least 2weeks before the test agent is introduced into the one or more fluidchannels of the device described herein. In some embodiments, the tumorspheroids are cultured in the presence of endothelial cells for at least1 week before the test agent is introduced into the one or more fluidchannels of the device described herein. This allows the tumor spheroidsand the endothelial cells to form a tumor tissue network.

Changes in the tumor cell spheroid culture which predict or demonstratea reduction in proliferation and/or dispersion of the tumor cellspheroids in the presence or absence of the test agent can be detectedusing known methods in the art, such as, chemical or physical methods,or a combination thereof. For example, a change in the tumor cellspheroid culture can be detected visually, e.g., using confocalmicroscopy imaging. The images obtained can be analyzed as described inAref et al. Integr Biol (Camb). 2013 February; 5(2):381-9, thedisclosure of which is incorporated by reference in its entirety. Inparticular, the paragraphs on pages 387-388 relating to imageacquisition and analysis (normalized dispersion, Δ/Δ₀, and normalizedcell number (N/N₀)) are incorporated by reference in their entirety. Insome embodiments, viability of the tumor cells is determined usingpropidium iodide, annexin V, or cellular ATP content, as disclosed byAmman et al. PLoS One 2014 Mar. 24; 9(3):e92511 and Zhu et al., CancerDiscov 2014 Apr; 4(4):452-65 (each incorporated herein by reference inits entirety.

In some embodiments, the change in the tumor cell spheroid culture is aclustering of immune cells around one or more tumor cell spheroids inthe culture. In some embodiments, the change in the tumor cell spheroidculture is a decrease in size and/or number of cells of one or moretumor cell spheroids in the culture.

In some embodiments, the change in the tumor cell culture is detectedchemically. For example, in some embodiments, the change in the tumorcell spheroid culture is determined by detection of the presence of abiological molecule secreted into the culture supernatant. In someembodiments, the biological molecule is a protein, carbohydrate, lipid,nucleic acid, metabolite, or a combination thereof. In some embodiments,the biological molecule is a chemokine or a cytokine. In someembodiments, the biological molecule is known to be associated withactivation of the immune system or otherwise an enhancement of theimmune response.

In some embodiments, the detected biological molecule(s) involves singlecell sequencing of T cell receptors on tumor spheroid associated CD4 andCD8 T cells that become activated in the device.

In some embodiments, a method of the invention comprises obtaining asample of tumor cell spheroid culture supernatant. In some embodiments,a method of the invention comprises detecting a secreted biologicalmolecule or a profile of secreted biological molecules, e.g., a cytokineprofile or chemokine profile, in the tumor cell spheroid culturesupernatant.

Methods for detecting secreted biological molecules are known in theart. In some embodiments, a multiplex profiling assay is used todetermine a profile of secreted biological molecules. For example, aBio-Plex® Multiplex Assay (BioRad) may be used. The Bio-Plex® MultiplexAssay is able to distinguish up to 100 different families ofcolor-coded, monodisperse polystyrene beads, each bearing a differenthomogeneous capture assay (but all using the same signal molecule) in asingle 50 μl sample. This high degree of multiplexing dramaticallyincreases the amount of useful information from rare or volume-limitedsamples. The Bio-Plex® assays are built around the well-known LuminexxMAP technology using a bead-based flow cytometric platform dedicated tomultiplex analysis Similar to ELISA, a majority of assays are designedaccording to a capture sandwich immunoassay format. Briefly, the captureantibody-coupled beads are first incubated with antigen standards orsamples for a specific time. The plate is then washed to remove unboundmaterials, followed by incubation with biotinylated detectionantibodies. After washing away the unbound biotinylated antibodies, thebeads are incubated with a reporter streptavidin-phycoerythrin conjugate(SA-PE). Following removal of excess SA-PE, the beads are passed throughthe array reader, which measures the fluorescence of the bound SA-PE.The substrate for the antibody sandwich is the bead. xMAP assays maycontain nonmagnetic or magnetic beads as substrates. Magnetic COOH beadsare unique in that they exhibit both fluorescent and magneticproperties. The beads are stained with a fluorescent dye formulationproprietary to Luminex. The staining process involves swelling the beadparticles in a dye containing solvent, which allows the dye molecules toinfuse into the coating or the polymer layer. Removal of the solvent ina subsequent step shrinks the beads and traps the dye molecules withinthe bead particles. The magnetite layer of the bead is one importantfeature that allows many of the newer assays to be automated withrobotic wash stations.

In some embodiments, the detected biological molecule(s) include one ormore of the following: Hu 6Ckine/CCL21, Hu BCA-1/CXCL13, Hu CTACK/CCL27,Hu ENA-78/CXCLS, Hu Eotaxin/CCL11, Hu Eotaxin-2/CCL24, HuEotaxin-3/CCL26, Hu Fractalkine/CX3CL1, Hu GCP-2/CXCL6, Hu GM-CSF, HuGro-a/CXCL1, Hu Gro-b/CXCL2, Hu I-309/CCL1, Hu IFN-g, Hu IL-10, HuIL-16, Hu IL-lb, Hu IL-2, Hu IL-4, Hu IL-6, Hu IL-8, Hu IP-10/CXCL10, HuI-TAC/CXCL11, Hu MCP-1/CCL2, Hu MCP-2/CCL8, Hu MCP-3/CCL7, HuMCP-4/CCL13, Hu MDC/CCL22, Hu MIF, Hu MIG/CXCL9, Hu MIP-1a/CCL3, HuMIP-1d/CCL15, Hu MIP-3a/CCL20, Hu MIP-3b/CCL19, Hu MPIF-1/CCL23, HuSCYB16/CXCL16, Hu SDF1a+b/CXCL12, Hu TARC/CCL17, Hu TECK/CCL25, and HuTNF-a.

In some embodiments, the detected biological molecule(s) include one ormore of the following: Hu Eotaxin, Hu FGF basic, Hu G-CSF, Hu GM-CSF, HuIFN-g, Hu IL-10, Hu IL-12 (p70), Hu IL-13, Hu IL-15, Hu IL-17, Hu IL-1b,Hu IL-1ra, Hu IL-2, Hu IL-4, Hu IL-5, Hu IL-6, Hu IL-7, Hu IL-8, HuIL-9, Hu IP-10, Hu MCP-1(MCAF), Hu MIP-1a, Hu MIP-1b, Hu PDGF-bb, HuRANTES, Hu TNF-a, and Hu VEGF. In some embodiments, the test agentinhibits epithelial-mesenchymal transition (EMT). In some embodiments,the test agent is a small molecule compound. In some embodiments, themethods described herein are used to screen a library of test agents,for example, a library of chemical compounds. In some embodiments, thetest agent comprises a nucleic acid molecule, for example, a DNAmolecule, an RNA molecule, or a DNA/RNA hybrid molecule,single-stranded, or double-stranded. In some embodiments, the test agentcomprises an RNAi agent, for example, an antisense-RNA, an siRNA, anshRNA, a snoRNA, a microRNA (miRNA), or a small temporal RNA (stRNA). Insome embodiments, the test agent comprises an aptamer. In someembodiments, the test agent comprises a protein or peptide. In someembodiments, the test agent comprises an antibody or an antigen-bindingantibody fragment, e.g., a F(ab′)2 fragment, a Fab fragment, a Fab′fragment, or an scFv fragment. In some embodiments, the antibody is asingle domain antibody. In some embodiments, the agent comprises aligand-or receptor-binding protein. In some embodiments, the agentcomprises a gene therapy vector.

In some embodiments, more primary tumor cell spheroids are cultured inthe presence of more than one agent, e.g., a first test agent and asecond test agent, optionally a third test agent, fourth agent, etc.

In some embodiments, a test agent is an anti-cancer agent. In someembodiments a test agent is a chemotherapeutic agent, animmunomodulatory agent, or radiation.

Exemplary chemotherapeutic agents include asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc. In some embodiments, a test agent is avinca alkaloid, e.g., vinblastine, vincristine, vindesine, vinorelbine.In some embodiments, a test agent is an alkylating agent, e.g.,cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide. Insome embodiments, a test agent is an antimetabolite, e.g., folic acidantagonists, pyrimidine analogs, purine analogs or adenosine deaminaseinhibitor, e.g., fludarabine. In some embodiments, a test agent is anmTOR inhibitor. In some embodiments, a test agent is a proteasomeinhibitor, e.g., aclacinomycin A, gliotoxin or bortezomib.

Exemplary immunomodulatory agents include immune activating agents orinhibitors of an immune checkpoint protein selected from the groupconsisting of: CTLA-4, PD-1, PDL-1, TIM3, LAG3, B7-H3 (CD276), B7-H4,4-1BB (CD137), OX40, ICOS, CD27, CD28, PDL-2, CD80, CD86, B7RP1, HVEM,BTLA, CD137L, OX40, CD70, CD40, CD40L, GAL9, A2aR, and VISTA. In someembodiments, the immune checkpoint inhibitor is a peptide, antibody,interfering RNA, or small molecule. In some embodiments, the immunecheckpoint inhibitor, e.g., inhibitor, is a monoclonal antibody, or anIg fusion protein. In some embodiments, the immune checkpoint inhibitoris an antibody or an antigen binding fragment thereof. In someembodiments, the immune checkpoint inhibitor an anti-PD-1 antibody.

In some embodiments, the immune checkpoint inhibitor inhibits PD1. Insome embodiments, the immune checkpoint inhibitor inhibits CTLA-4. Insome embodiments, the immune checkpoint inhibitor inhibits TIM-3. Insome embodiments, the immune checkpoint inhibitor inhibits LAG-3. Insome embodiments, the immune checkpoint inhibitor inhibits VISTA.

In some embodiments, a combination of test agents are tested in the cellculture. In some embodiments, the combination of immune checkpointinhibitors includes a PD1 inhibitor and a CTLA-4 inhibitor. In someembodiments, the combination of immune checkpoint inhibitors includes aPD1 inhibitor and a TIM-3 inhibitor. In some embodiments, thecombination of immune checkpoint inhibitors includes a PD1 inhibitor anda LAG-3 inhibitor. In some embodiments, the combination of immunecheckpoint inhibitors includes a CTLA-4 inhibitor and a TIM-3 inhibitor.In some embodiments, the combination of immune checkpoint inhibitorsincludes a CTLA-4 inhibitor and a LAG-3 inhibitor. In some embodiments,the combination of immune checkpoint inhibitors includes a TIM-3inhibitor and a LAG-3 inhibitor. In some embodiments, the combination ofimmune checkpoint inhibitors includes a PD1 inhibitor , a CTLA-4inhibitor, and a TIM-3 inhibitor. In some embodiments, the combinationof immune checkpoint inhibitors includes a PD1 inhibitor, a CTLA-4inhibitor , and a LAG-3 inhibitor. In some embodiments, the combinationof immune checkpoint inhibitors includes a PD1 inhibitor, a TIM-3inhibitor, and a LAG-3 inhibitor. In some embodiments, the combinationof immune checkpoint inhibitors includes a CTLA-4 inhibitor, a TIM-3inhibitor , and a LAG-3 inhibitor.

In some embodiments, an immune activating agent is a CD28 antagonist,e.g., an anti-CD28 antibody.

In some embodiments, a test agent is a small molecule inhibitor, e.g., aTBK1 inhibitor, a MEK inhibitor, a FAK inhibitor, a BRD/BET inhibitor, aCDK 4/6 inhibitor, an HDAC inhibitor, a DNMT inhibitor (orhypomethylating agent), a MET inhibitor, an EGFR inhibitor, or a BRAFinhibitor. In some embodiments, a test agent is a kinase inhibitor,e.g., a

TBK1 inhibitor, a MEK inhibitor, a FAK inhibitor, or a CDK 4/6inhibitor.

In some embodiments, the proliferation and/or dispersion of the tumorcell spheroids is reduced in the presence of the test agent as comparedto the absence of the agent, indicating that the agent can be used totreat cancer. In some embodiments, the proliferation and/or dispersionof the tumor cell spheroids is reduced in the presence of the test agentas compared to the absence of the agent by at least 10%, 25%, 50%, 75%,90%, 95% or 100%.

In some embodiments, the size and/or number of cells of the tumor cellspheroids is reduced in the presence of the test agent as compared tothe absence of the agent, indicating that the agent can be used to treatcancer. In some embodiments, the size and/or number of cells of thetumor cell spheroids is reduced in the presence of the test agent ascompared to the absence of the agent by at least 10%, 25%, 50%, 75%,90%, 95% or 100%.

In some embodiments, a change in the secretion of a biological moleculeinto the cell culture supernatant in the presence of the test agent ascompared to the absence of the agent is indicative that the agent can beused to treat cancer. In some embodiments, the secretion of a biologicalmolecule into the cell culture supernatant, e.g., a change which isindicative of immune activation, is increased in the presence of thetest agent as compared to the absence of the agent by at least 10%, 25%,50%, 75%, 90%, 95% or 100%, indicating that the agent can be used totreat cancer. In some embodiments, the secretion of a biologicalmolecule into the cell culture supernatant, e.g., a change which isindicative of immune suppression, is reduced in the presence of the testagent as compared to the absence of the agent by at least 10%, 25%, 50%,75%, 90%, 95% or 100%, indicating that the agent can be used to treatcancer.

In some embodiments, cells can be liberated from the cell culturedevice, e.g., by collagenase. The liberated cells can be subjected toanalyses such as, for example, flow cytometry, immunofluorescence, amongothers.

In some aspects, a subject can be treated with an agent identified asuseful for treating cancer according to a method described herein. Forexample, in some embodiments, the present disclosure provides a methodfor treating cancer in a subject, the method comprising: a) obtaining atumor sample from the subject; b) identifying an agent that can be usedto treat cancer in the subject according to a method described herein;and c) administering the agent to the subject.

The present invention is further illustrated by the following Example,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1

Materials and Methods

Freezing Medium:

1 mL DMEM (w/10% FBS, 1× Antibiotic/Antimycotic, 1× L-Glutamine)

8 mL FBS

1 mL DMSO

Sterile Filter with 0.22 um syringe filter if all components are notsterile

Matrigel

BD Biosciences ‘BD Matrigel™ Matrix Basement Membrane’ (Part number354234). NB: Thaw completely overnight @ 4° C. the aliquot which youwill be using; always keep cold, using pre-cooled tubes, tips, andsyringes (it gels at temps slightly above Room Temp).

Collagen Type I

BD Biosciences

Preparation of 1,000× Insulin(10 mg/mL):

50 mg of powder (one bottle)+5 mL of 6 μM HCl

Recipe for 1× Collagenase Buffer Solution:

DMEM/F12 or RPMI with 15 mM HEPES containing:

FBS (5%)

Insulin (10 ng/mL)

EGF(10 ng/mL)

Hydrocortisone(10 μg/mL)

Pen/Strep/Fung(1×)

Collagenase(0.5 mg/mL)

Preparation of 500 mLs of Collagenase Buffer Solution (for OvernightDissociation)

DMEM/F12 with 15 mM HEPES: 500 mL

Calf serum or FBS: 25 mL (5%)

1,000× Insulin: 500 μl(final con: 10 ng/mL)

Pen/Strep/Fung(100×): 5 mL

Hydrocortisone (final con: 10 μg/mL):

Collagenase (final con: 0.5 mg/mL)

Generation of Spheroids

-   -   1. To generate spheroids, the human primary tissues (fresh        mesothelioma or PDX samples) were collected in media (RPMI) with        10% FBS.    -   2. Mince samples into tiny pieces on ice (approximately 1 mm).        Then transfer the minced samples back on ice in a 50 mL conical        tube containing 20 mL media. Spin tissue down at 800 rpm for 2        min and remove supernatant.    -   3. Collagenase the samples in collagenase buffer solution (see        materials). Time of incubation ranged from 2 hours (fresh        mesothelioma sample) or up to 12 hours (PDX breast sample) at 37        degrees Celsius (on a rotator in the dark if available)        incubator.    -   4. Spin down the solution at 1000 rpm for 5 min and discard the        supernatant.    -   5. Wash the samples 2 times: by adding 30 mL of PBS,        re-suspending the samples, and then spin down the samples at        1000 rpm for 5 min, discarding the supernatant.    -   6. Re-suspended pellet in 20 mL fresh media, pipette up and down        for 2-3 minutes (mix them well by 25 mL surgical pipette).    -   7. Individual spheroids were sieved via 100 μm and 40 μm cell        strainers to yield spheroids 40-100 μm in diameter.    -   8. Harvest the spheroids from 40 μm strainer with 10 mL fresh        media and centrifuged to separate them from the supernatant.        Tissue Freezing Procedure:    -   1. Freezing was performed from either directly minced tissue        (PDX was minced and frozen in 90% FBS and 10% DMSO at −80° C.)        or after tumor spheroids were harvested.    -   2. For freezing of tumor spheroids, spheroids from step 8 were        centrifuged at 1000 rpm for 3 min.    -   3. Aspirate Medium.    -   4. Resuspend spheroids in 1 mL freezing medium (see appendix)        per ampule to be frozen.    -   5. Aliquot samples into labeled ampules and put at −80° C.        overnight.    -   6. Transfer cells the next day into liquid nitrogen for storage.        Thawing Procedure:    -   1. Remove tissue spheroid samples from liquid nitrogen and        immediately place in 37° C. water bath to thaw quickly.    -   2. Resuspend spheroids from each vial in 35 mL DMEM Media with        10% FBS, 1× Antibiotic/Antimycotic, 1× L-Glutamine.    -   3. Spin down spheroids and re-suspend in collagen or matrigel        for injection into the devices.        Results        Culture Device Fabrication and Tissue Implantation in 3D Matrix

One embodiment of a three dimensional microfluidic culture device wasfabricated according to the disclosures of Aref et al., IntegrativeBiology 2013; PMID 23172153 and US published patent application US2014-0057311 A1 (each herein incorporated by reference in its entirety).Mesothelioma spheroids were generated from primary tumor tissue, frozen,thawed, and implanted into the device, by the methods described herein.Spheroids were visualized in the device by immunofluorescence confocalmicroscopy, as shown in FIG. 1. In another experiment, spheroids wereco-cultured with human umbilical vein endothelial cells (HUVECs) in thedevice, as shown in FIG. 2. FIG. 3 shows confocal microscopy picturesdepicting mesothelioma cell dissemination in 3 dimensions over a periodof 48 hours after implantation into the device.

References

1. Tentler J J, Tan A C, Weekes C D, Jimeno A, Leong S, Pitts T M, etal. Patient-derived tumour xenografts as models for oncology drugdevelopment. Nature reviews Clinical oncology. 2012; 9(6):338-50. EpubApr. 18, 2012. doi: 10.1038/nrclinonc.2012.61. PubMed PMID: 22508028;PubMed Central PMCID: PMC3928688.2. Aref A R, Huang R Y, Yu W, Chua K N, Sun W, Tu T Y, et al. Screeningtherapeutic EMT blocking agents in a three-dimensional microenvironment.Integrative biology: quantitative biosciences from nano to macro. 2013;5(2):381-9. Epub Nov. 11, 2012. doi: 10.1039/c2ib20209c. PubMed PMID:23172153.3. Zhu Z, Aref A R, Cohoon T J, Barbie T U, Imamura Y, Yang S, et al.Inhibition of KRAS-Driven Tumorigenicity by Interruption of an AutocrineCytokine Circuit. Cancer discovery. 2014; 4(4):452-65. Epub Jan. 22,2014. doi: 10.1158/2159-8290.CD-13-0646. PubMed PMID: 24444711.4. Yu M, Bardia A, Aceto N, Bersani F, Madden M W, Donaldson M C, et al.Cancer therapy. Ex vivo culture of circulating breast tumor cells forindividualized testing of drug susceptibility. Science. 2014;345(6193):216-20. Epub Jul. 12, 2014. doi: 10.1126/science.1253533.PubMed PMID: 25013076.5. Gao D, Vela I, Sboner A, Iaquinta P J, Karthaus W R, Gopalan A, etal. Organoid cultures derived from patients with advanced prostatecancer. Cell. 2014; 159(1):176-87. Epub Sep. 10, 2014.doi:10.1016/j.cell.2014.08.016. PubMed PMID: 25201530.6. Gerdes M J, Sevinsky C J, Sood A, Adak S, Bello M O, Bordwell A, etal. Highly multiplexed single-cell analysis of formalin-fixed,paraffin-embedded cancer tissue. Proceedings of the National Academy ofSciences of the United States of America. 2013; 110(29):11982-7. EpubJul. 3, 2013. doi: 10.1073/pnas.1300136110. PubMed PMID: 23818604;PubMed Central PMCID: PMC3718135.7. Yao Z, Fenoglio S, Gao D C, Camiolo M, Stiles B, Lindsted T, et al.TGF-beta IL-6 axis mediates selective and adaptive mechanisms ofresistance to molecular targeted therapy in lung cancer. Proceedings ofthe National Academy of Sciences of the United States of America. 2010;107(35):15535-40. Epub Aug. 18, 2010. doi: 10.1073/pnas.1009472107.PubMed PMID: 20713723; PubMed Central PMCID: PMC2932568.8. Seguin L, Kato S, Franovic A, Camargo M F, Lesperance J, Elliott K C,et al. An integrin beta(3)-KRASRalB complex drives tumour stemness andresistance to EGFR inhibition. Nature cell biology. 2014; 16(5):457-68.Epub Apr. 22, 2014. doi: 10.1038/ncb2953. PubMed PMID: 24747441.

Example 2 3D Culture of Spheroids

The overview of an exemplary method of 3D culture of spheroids is shownin Table 1.

TABLE 1 Day −1 Device fabrication/preparation* Day 1 Sample collection,collagenase digestion, spheroid filtration/collection Day 2 Spheroidcollection/harvest, resuspension in collagen, loading device Collagenpolymerization Addition of culture media Addition of endothelial cellsDay 3 Treatment with test agent(s) Day 5-6 Collect conditioned media,e.g., for identification of secreted biological molecule(s), e.g.,multiplex cytokine analysis Day 6+ Monitor cell growth, fix cells,immunostaining, RNAseq, flow cytometry

On Day 1, tumor was collected in an operating room and placed in asterile 15 mL conical tube bathed with culture media (10% FBS with DMEMor RPMI) and stored on ice. The media was aspirated in a sterile hoodand the tumor specimen gently placed in a normal 10 cm culture dish onice. Using a scalpel, the tumor was minced into smaller pieces. A samplecan optionally be exposed to strong collagenase treatment to generatesingle cells that can be submitted directly for flow cytometry. To the10 cm dish containing the minced tumor was added 10 mL of collagenasesolution (10 mL media, 150 μL HEPES (15 mM final concentration), 100 μLcollagenase (100 U/μL stock, 1 unit/μL final concentration)). The mincedtumor and collagenase mixture was subsequently transferred to a 50 mLconical tube, and then transferred to a fresh low-attachment 10 cm dish.The low-attachment dish comprising the sample was incubated at 37° C.for 30-60 minutes while checking every 15-30 minutes, as the duration oftime required varies from specimen to specimen. The intended outcome ofthis step is to generate multicellular spheroids via a limitedcollagenase digestion, so the sample should not be comprised entirely ofsingle cells. After the 45-60 minute incubation period, 10 mL of freshmedia was added to the dish containing the tumor sample and the contents(˜20 mL) transferred to a 50 mL conical tube. The solid tumor and tumorspheroids were then pelleted via centrifugation at 1200-1300 rpm for 4-5minutes. The resulting supernatant was transferred to a new tube forfuture re-use of the collagenase solution, and the pellet wasresuspended in 10-20 mL media (e.g., 10% FBS/DMEM) using a 25 mL pipet.The media containing the solid tumor and tumor spheroids was passed overa 100 μm filter that was resting on a 50 mL conical tube. The filter,which had captured residual tumor and spheroids greater than 100 μm, wasinverted, rinsed with residual collagenase-media solution, transferredto “Dish 1,” and returned to the 37° C. incubator. This step can beperformed to recover additional spheroids on Day 2 after continued,limited collagenase digestion. The flow-through from the 100 μm filterwas passed over a 40 μm filter that was resting on a fresh 50 mL conicaltube. The 40 μm filter was inverted and the cells were recovered bypassing 10 mL media (10% FBS/DMEM) over the filter and collecting cellsinto a 10 cm low attachment petri dish (“Dish 2,” 40-100 μm). Theflow-through from the 40 μm filter was placed in a separate 10 cm dish(“Dish 3,” <40 μm, mostly single cells), and returned to 37° C.overnight. Spheroids can be immediately loaded into the 3D culturedevices or kept in culture overnight.

On Day 2, the spheroids in Dish #2 were examined under a microscope. Acollagen solution (150 μL rat tail collagen (type I), 20 μL 10× PBS, 7μL 1 N NaOH, 23 μL sterile H₂O) was prepared on ice. The media andspheroids from Dish #2 were collected and the spheroids pelleted at 1100rpm for 2 minutes. The media was aspirated and the 15 mL conical tubescontaining cell pellets were placed on ice. The cell pellets wereresuspended on ice via the addition of 200 μL (or more as needed) ofcollagen mixture. During this procedure, care was taken to limit theamount of time the pellet was not on ice. Approximately 15-20 μL ofspheroid-collagen mixture per device was added at either end of the gelchannel. The 3D devices were placed in humidified, sterile containersfor 45-60 min at 37° C. After approximately 1 hour, ˜200 μL of theappropriate culture media were added to hydrate the devices and providea nutrition and growth factor source to the spheroids, and the deviceswere returned to 37° C. incubation. While the polymerizedcollagen-spheroid mixture was being hydrated with media, HUVEC weretrypsinized, cells were pelleted and resuspended in 50-100 μL of EGM2media and loaded to the LEFT media channel. Alternatively, spheroids canbe grown in the absence of HUVEC.

On Day 3, media was aspirated from the device and ˜200 μL media (1:1mixture of 10% FBS-DMEM and EGM2 supplemented with growth factors perprotocol) with or without treatment (e.g., anti-PD-1 monoclonal Ab) wereadded to each device. The devices were subsequently placed inhumidified, sterile containers, and returned to 37° C. incubation.

On Day 6 (or 48-72 hours post-treatment), conditioned media wascollected for cytokine analysis. Light microscopy was performed andimages obtained to characterize and document spheroid dispersal and/orputative immune response, which is commonly characterized bynon-pigmented cells surrounding the brown-pigmented melanoma spheroids.Media was collected from each device by aspiration using a 200 μL pipet,transferred to sterile 1.5 mL Eppendorf tubes, and frozen at −80° C.prior to submission to CMOP for multiplex cytokine/chemokine analysis.Also on Day 6, the cells present in the 3D culture device followingtreatment were characterized by multiple methods.

For flow cytometry, RNA sequencing analysis, or T-cell receptor (TCR)sequencing the cells were removed using collagenase (150 μL per device)after media had been removed. The mixture was then incubated for 20-25minutes until the spheroids began to demonstrate movement within the 3Ddevice by light microscopy. The cells in collagenase were aspirated intoa 15 mL conical tube containing 4.0 mL of sterile 1× PBS and spun downfor 5 minutes at 1100 rpm. The PBS wash was gently aspirated and thepellet resuspended in 1.5 mL 10% FBS-DMEM for flow cytometry. In someinstances, genomic DNA is prepared from flow-sorted T lymphocytes forTCR sequencing using the ImmunoSeq platform from AdaptiveBiotechnologies to evaluate T-cell receptor clonality. Clonality can beassessed in spheroids relative to bulk tumor, and in response to immunecheckpoint blockade to evaluate for increased TCR clonality (indicatingexpansion of tumor-reactive T cell clones). For RNA sequencing, theresuspension can be performed using 1.5 mL 10% FBS-DMEM, PBS, or acompatible lysis buffer.

For direct immunofluorescence analysis, a fixing step was firstperformed after media had been removed. In this step, 200 μL of 4%paraformaldehyde was added to each device via gel and side ports andincubated for 15 minutes at room temp. Following the incubation period,paraformaldehyde was removed and 200 μL of 0.1% Triton X-100 was addedfor permeabilization and allowed to incubate for 10 minutes at roomtemperature. Triton X-100 was then removed and the device washed with 1×PBS 2-3 times. Next, 200 μL, of 5% FBS in PBS was added to each devicefollowed by a 30-60 minute incubation period at room temp. Anappropriate dilution of primary antibody in 5% FBS-PBS was added andfollowed by overnight incubation at 4° C. After the incubation period,primary antibody was removed and washed with 1× PBS 2-3 times. Thesecondary antibody was added as 1:500 Alexa-Fluor antibody in 5% FBS-PBSand incubated for 2-4 hours at room temperature, with care taken toavoid or minimize sample exposure to light. The secondary antibody wasthen removed and washed with 1× PBS 2-3 times. Approximately 200 μL PBSwas kept in device as fixed/stained devices can be directly visualizedby fluorescence microscopy or stored at 4° C. until they are visualized.

Example 3

Immunotherapy with PD-1 blockade is associated with significant activityin patients with metastatic melanoma, but durable responses are onlyobserved in a limited number of patients. To date there are no provenbiomarkers or patient characteristics that reliably predict response toimmune checkpoint inhibitors. PD-L1 expression predicts response toanti-PD-1/PD-L1 antibodies in some, but not all patients, and isincreasingly recognized as an imperfect marker of activity. As biopsiesfrom patients who have responded to these agents often demonstrate thepresence of an inflammatory infiltrate within the tumor, and geneexpression profiling studies have confirmed upregulation ofpro-inflammatory cytokines and chemokines within tumors following PD-1blockade, there is increasing interest in understanding the role of thetumor microenvironment in the response to immune checkpoint inhibition.Unfortunately, most approaches to evaluate the tumor microenvironmentrely on fixed tissue from biopsies, which precludes dynamic evaluationof features associated with response.

A microfluidic cell culture technology has been previously used to studydrug sensitivity of tumor cell line spheroids and was also shown tosupport the growth of primary human tumor specimens. The technologyrecapitulates the tumor microenvironment, incorporating a modelextracellular matrix (ECM), enabling cell-cell interactions that reflectthe endothelial and/or immune-cancer cell interface, and allowingcontrolled analysis of growth factor and cytokine mediated effects. Theadvantages of this system provided a basis for its use in examining theconsequences of PD-1 blockade ex vivo. In some embodiments, melanomaserved as the cancer model as it exhibits a relatively high responserate to anti-PD1 antibodies and also produces melanocyte pigments thatfunction as tumor cell-specific markers.

In spheroids derived from multiple different melanoma resectionspecimens, there was an observed immune response to anti-PD1(pembrolizumab 250 μg/mL) exposure in the absence (FIG. 4A) and presence(FIG. 4B) of anti-CD28 co-stimulation. As the spheroids and co-culturedendothelial cells are bathed in media, the conditioned media wasextracted from the device and luminex cytokine profiling was performed.There was an observed pronounced up-regulation of multiple cytokinesspecifically following αPD1 exposure, and certain cytokines (e.g.,IP10), which were induced by the combination with anti-CD28 (FIG. 5).Together, these findings demonstrated the ability of this system tocapture an immune response to PD1 blockade ex vivo, and quantify uniquecytokine profiles.

Additionally, although it was demonstrable by flow cytometry of melanomatumor spheroids at baseline that they consisted of up to 20% immunecells, including T cells, dendritic cells, monocytes (not shown), thevalue of extracting cells from the device and repeating flow cytometrypost-treatment was realized. By repeating collagenase digestion withinthe device and washing cells in PBS, conditions have been developed thatenable repeat immune profiling. Interestingly, following anti-PD1 orespecially anti-PD1/anti-CD28 treatment, there was an observed increasein markers of early CD8 T cell activation such as CD69 (FIG. 6A), whichwas accompanied by an increase in specific immune checkpoint receptorssuch as TIM3 (FIG. 6B). Thus, in addition to characterizing differentialsecreted cytokine profiles, the microfluidic culture system provides thecapacity to study the impact of checkpoint blockade on the activation ofT cells and other immune populations in real time on primary patienttumor specimens.

1. Lo J A, Fisher D E. The melanoma revolution: from UV carcinogenesisto a new era in therapeutics. Science. 2014; 346(6212):945-9. Epub Nov.22, 2014. doi: 10.1126/science.1253735. PubMed PMID: 25414302.

2. Carbognin L, Pilotto S, Milella M, Vaccaro V, Brunelli M, Calio A,Cuppone F, Sperduti I, Giannarelli D, Chilosi M, Bronte V, Scarpa A,Bria E, Tortora G. Differential Activity of Nivolumab, Pembrolizumab andMPDL3280A according to the Tumor Expression of Programmed Death-Ligand-1(PD-L1): Sensitivity Analysis of Trials in Melanoma, Lung andGenitourinary Cancers. PLoS One. 2015; 10(6):e0130142. doi:10.1371/journal.pone.0130142. PubMed PMID: 26086854; PMCID: 4472786.3. Taube J M, Young G D, McMiller T L, Chen S, Salas J T, Pritchard T S,Xu H, Meeker A K, Fan J, Cheadle C, Berger A E, Pardoll D M, Topalian SL. Differential Expression of Immune-Regulatory Genes Associated withPD-L1 Display in Melanoma: Implications for PD-1 Pathway Blockade. ClinCancer Res. 2015; 21(17):3969-76. doi: 10.1158/1078-0432.CCR-15-0244.PubMed PMID: 25944800.4. Aref A R, Huang R Y, Yu W, Chua K N, Sun W, Tu T Y, Bai J, Sim W J,Zervantonakis I K, Thiery J P, Kamm R D. Screening therapeutic EMTblocking agents in a three-dimensional microenvironment. Integr Biol(Camb). 2013; 5(2):381-9. doi: 10.1039/c2ib20209c. PubMed PMID:23172153; PMCID: 4039387.5. Zhu Z, Aref A R, Cohoon T J, Barbie T U, Imamura Y, Yang S, Moody SE, Shen R R, Schinzel A C, Thai T C, Reibel J B, Tamayo P, Godfrey J T,Qian Z R, Page A N, Maciag K, Chan E M, Silkworth W, Labowsky M T,Rozhansky L, Mesirov J P, Gillanders W E, Ogino S, Hacohen N, Gaudet S,Eck M J, Engelman J A, Corcoran R B, Wong K K, Hahn W C, Barbie D A.Inhibition of KRAS-driven tumorigenicity by interruption of an autocrinecytokine circuit. Cancer Discov. 2014; 4(4):452-65. doi:10.1158/2159-8290.CD-13-0646. PubMed PMID: 24444711; PMCID: 3980023.

We claim:
 1. A method for culturing patient-derived tumor cell spheroidsin a three-dimensional microfluidic device, the method comprising:mincing a primary tumor sample in a medium supplemented with serum;treating the minced primary tumor sample with a composition comprisingan enzyme; collecting tumor spheroids having a diameter of 10 μm to 500μm from the enzyme treated sample; suspending the tumor spheroids inbiocompatible gel; and culturing the tumor spheroids in a threedimensional microfluidic device.
 2. The method of claim 1, wherein theminced primary tumor sample is frozen in a medium supplemented withserum and thawed prior to treating with the composition comprising theenzyme.
 3. The method of claim 1, wherein the collected tumor spheroidsare frozen in a freezing medium and then thawed before suspending in thebiocompatible gel.
 4. The method of claim 1, wherein the minced primarytumor sample comprises tumor pieces in the size of about 1 mm.
 5. Themethod of claim 1, wherein the tumor spheroids having a diameter of 10μm to 500 μm are collected from the enzyme mix treated sample with theuse of a sieve.
 6. The method of claim 1, wherein the tumor spheroidshaving a diameter of 10 μm to 500 μm are collected by sieving the enzymemix treated sample via 500 μm and 10 μm cell strainers to yield tumorspheroids having a diameter of 10 μm to 500 μm.
 7. The method of claim1, wherein the enzyme is collagenase.
 8. The method of claim 1, whereinthe composition comprising the enzyme comprises a serum-supplementedculture medium, insulin, a corticosteroid, an antibiotic, collagenaseand optionally a growth factor.
 9. The method of claim 1, wherein theminced primary tumor sample is treated with the composition comprisingthe enzyme in an amount or for a time sufficient yield a partialdigestion of the minced primary tumor sample.
 10. The method of claim 1,wherein the minced primary tumor sample is treated with the compositioncomprising the enzyme for 30 minutes to 15 hours at a temperature of 25°C. to 39° C.
 11. The method of claim 1, wherein the three dimensionalmicrofluidic device comprises: one or more fluid channels flanked by oneor more gel cage regions, wherein the one or more gel cage regionscomprises the biocompatible gel in which the tumor spheroids areembedded, and wherein the device recapitulates in vivo tumormicroenvironment.
 12. The method of claim 1, wherein the threedimensional microfluidic device comprises: a substrate comprised of anoptically transparent material and further comprising i) one or morefluid channels; ii) one or more fluid channel inlets; iii) one or morefluid channel outlets; iv) one or more gel cage regions; and v) aplurality of posts; wherein all or a portion of each gel cage region isflanked by all or a portion of one or more fluid channels, therebycreating one or more gel cage region-fluid channel interface regions;each gel cage region comprises at least one row of posts which forms thegel cage region; and the one or more gel cage region has a height of atleast 500 μm.
 13. The method of claim 12, wherein the one or more gelcage region has a height sufficient for at least 200-1000 μm above thetumor cell spheroids.
 14. The method of claim 12, wherein the gel cageregion has a cuboidal shape.
 15. The method of claim 1, wherein thetumor spheroids are cultured in the presence and absence of a testagent.
 16. The method of claim 1, further comprising detecting a changein the tumor cell spheroid culture indicative of a condition that islikely to reduce proliferation and/or dispersion of the tumor cellspheroids in the presence of the first test agent as compared to theabsence of the first test agent.